The insulin-like growth factor 1 (IGF-1) receptor is an essential regulator of cell growth and transformation. IGF-1 and IGF-2, via the IGF-1 receptor, are potent breast cancer cell mitogens that promote the tumorigenic potential of cancer cells. Antagonism of the IGFs and/or blockade of IGF-1R signaling are therefore, therapeutically important targets for the development of anti-cancer drugs. The objective of this proposal is to develop reagents that block IGF-1 receptor signaling, using a unique approach. The insulin-like growth factor binding proteins (IGFBPs) bind IGF-1 and IGF-2 with higher affinities than the IGF-1 receptor and serve to both protect the IGFs from degradation and reduce their delivery to the IGF-1 receptor. Our hypothesis is that blockade of IGF-1 receptor activity can be accomplished by developing antagonists of the IGFs based on the structure of the IGFBPs. Specifically, we propose to generate peptide antagonists having the structural features of the IGF binding domain on the IGFBPs. Such analogs should serve to antagonize the binding of IGF-1 and IGF-2 to the IGF-1 receptor and thus abrogate their effects. Because insulin and IGF-1 have similar receptor binding domains and specificities, antagonists based on the structure of the IGF-1 binding domain on the IGF-1 receptor would have the complication of antagonizing insulin: insulin receptor interactions. In strong support of our hypothesis, our preliminary studies show that recombinant human IGFBP-2 (rhIGFBP-2) inhibits basal (IGF autocrine loop) and IGF-1 stimulated MCF-7 cell proliferation. The goal of AIM 1 is to define the IGF binding site on rhIGFBP-2 using the combined approaches of photoaffinity labeling and mass spectrometry. For this purpose, IGF-1 derivatized with a photoactivatable group within its IGFBP binding domain will be synthesized. This will allow the precise identification of the site of interaction between rhIGFBP-2 and IGF-1. The goal of AIM 2 is to synthesize peptides and generate small molecular weight rhIGFBP-2 fragments which retain a high affinity for IGF-1. These peptides will be based on the structure of the IGF binding domain determined in AIM 1, as well as modifications of the IGFBP binding site on IGF-1. The combined approaches will lead to the development of therapeutic agents aimed at reducing the tumorigenic effects of the IGFs in breast cancer.